Thousands of young males under the age of 20 are diagnosed with cancer each year. The overall cure rates of these cancer patients are approaching 80%; therefore, the number of childhood cancer survivors is increasing over time. It is well documented that cancer treatments may result in temporary, long-term, or permanent gonadal failure in male cancer survivors. Certain benign hematological disorders, such as myelodysplasia, sickle cell disease, aplastic anemia, thalassemia major, and Fanconi anemia, and severe autoimmune diseases unresponsive to immunosuppressive therapy, such as juvenile idiopathic arthritis, juvenile systemic lupus erythematosus, systemic sclerosis and immune cytopenias, necessitate administration of high dose chemotherapy that may also impact testicles. In immature boys, in particular, storing testicular tissue prior to such cytotoxic treatments for future replantation could be an option. This proposal specifically addresses cryopreservation of pediatric testes. This male reproductive organ is contained within the scrotum, and is responsible for the production of sperm and the male hormone, testosterone that is pivotal for the maintenance of male sexual characteristics. As such, injury to the testes can render a young male infertile and needing administration of exogenous testosterone. Klinefelter?s syndrome patients would also benefit. Klinefelter?s syndrome the most common (1:500 new male born) genetic reason for male infertility, suffer from progressive germ cell loss and testis fibrosis with onset of puberty. We propose to remedy this by cryopreservation of testes that can be used for restoration of sexual function, fertility and hormonal balance in males before they are damaged. A single testis from each patient would be cryopreserved. This would permit either replantation of the patient?s intact testis, implantation of viable spermatogonial stem cells in the remaining testis, or ectopic implantation of functional spermatogonial stem cells. Each strategy would be capable of completing spermatogenesis and provide testosterone in a regulated way. We propose a novel innovative approach using ice-free vitrification to cryopreserve pediatric testes employing a young rat model. No animals will be sacrificed because we are using heterozygous and wild type culls from breeding colonies post-mortem. In this Phase I SBIR proposal in vitro methods and histology will be used to assess outcomes in three specific aims. Feasibility for progression to a Phase II SBIR grant proposal will be demonstrated by achievement of >80% cell viability by 3 methods and <20% apoptotic cells after 3 months of storage in vapor phase nitrogen. We have already assembled a world-class team of experts in surgery and reproductive medicine to guide the Company in Phase II and Phase III commercialization efforts.